Laminins, derivatives, and compositions including same and methods for their therapeutic use

ABSTRACT

In various embodiments, the present disclosure provides a method of treating a subject using laminin or a composition that includes laminin. In one embodiment, the method is used to enhance muscle regeneration, maintenance, or repair in a subject. In another embodiment, the method is used to promote wound healing. The method, in yet another embodiment, is used to prevent or reduce muscle damage or injury. In specific implementations of these methods, the laminin or composition that includes laminin is administered in a therapeutically effective amount. In some implementations, the laminin is a complete laminin protein. In other implementations, the laminin is a laminin fragment, a laminin derivative, or a laminin analogue.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of, and incorporates by reference,U.S. Provisional Patent Application No. 60/998,320, filed Oct. 9, 2007.This application incorporates by reference International PatentApplication No. PCT/US08/78459, filed Oct. 1, 2008.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with United States Government support undergrants from the National Institutes of Health (NIH), National Center forResearch Resources, Grant Nos. NCRR P20 RR018751-01, P20 RR15581-04;National Institute of Arthritis and Musculoskeletal and Skin Diseases,Grant No. NIAMS R01AR053697-01; and National Institute of NeurologicalDisorders and Stroke, Grant No. NINDS R21NS058429-01. The United StatesGovernment has certain rights in the invention.

FIELD

The present disclosure relates to a method of providing therapeuticbenefit to a subject by administering to the subject a laminin or acomposition that includes laminin. In a particular embodiment, thepresent disclosure provides a method of enhancing muscle regeneration,such as to treat muscular dystrophy, in a subject by administeringlaminin or a laminin composition.

BACKGROUND

Adult skeletal muscle exhibits a remarkable ability to repair andregenerate after trauma or injury. The regenerative capacity of skeletalmuscle is due to a reservoir of satellite cells located under the basallamina and in close proximity to the myofiber sarcolemma. These cellsremain quiescent in healthy uninjured muscle, but are rapidly activatedin response to muscle damage, exercise, or disease.

Upon activation, satellite cells proliferate and differentiate down themyogenic pathway and are able to repair damaged muscle. Models suggest asubpopulation of satellite cells remain as stem cells to replaceactivated cells that have progressed down the myogenic lineage pathway.During the activation period, satellite cells express the transcriptionfactors Pax3, Pax7, MyoD, myogenin, and MRF4 as they progress through adevelopmental program towards muscle repair.

Muscular dystrophy is a term used to refer to a group of geneticdisorders that lead to progressive muscle weakness. Muscular dystrophycan result in skeletal muscle weakness and defects in skeletal muscleproteins, leading to a variety of impaired physiological functions. Nosatisfactory treatment of muscular dystrophy exists. Existing treatmentstypically focus on ameliorating the effects of the disease and improvingthe patient's quality of life, such as through physical therapy orthrough the provision of orthopedic devices.

Mutated genes associated with muscular dystrophy are responsible forencoding a number of proteins associated with the costameric proteinnetwork. Such proteins include laminin-2, collagen, dystroglycan,integrins, caveolin-3, ankyrin, dystrophin, α-dystrobrevin, vinculin,plectin, BPAG1b, muscle LIM protein, desmin, actinin-associated LIMprotein, α-actin, titin, telethonin, cypher, myotilin, and thesarcoglycan/sarcospan complex.

The most common form of muscular dystrophy, Duchenne muscular dystrophy,is caused by a mutation in the gene responsible for production ofdystrophin. Dystrophin is a protein involved in binding cells to theextracellular matrix, including the basement membrane. Congenitalmuscular dystrophies are caused by gene mutations affecting theproduction of other costameric proteins. For example, in populations ofEuropean descent, the most prevalent congenital muscular dystrophy iscaused by a mutation resulting in a lack of α7β1 integrin expression.Like dystrophin, α7β1 integrin is involved in binding cells to theextracellular matrix.

To some extent, a defect in the gene encoding for one of dystrophin orα7β1 integrin is often compensated for by enhanced expression of theother, or another costameric protein, such as utrophin (an analog ofdystrophin). Dystrophin, α7β1 integrin, and utrophin all serve asreceptors for laminin, which serves as the link to the extracellularmatrix. Defective production of laminin-2 itself gives rise tomerosin-deficient congenital muscular dystrophy (MCMD) or congenitalmuscular dystrophy type 1A (MDC1A).

Laminin is a major component of the basement membrane. At least fifteenlaminin protein trimers have been identified, each a heterotrimerincluding an α, β, and γ chain. Laminin is associated with a number ofphysiological functions, including cell attachment, gene expression,tyrosine phosphorylation of proteins, cell differentiation, as well ascell shape and movement. Laminin is known to bind to cell membranesthrough integrin receptors. In addition, laminin-2 binds toα-dystroglycan as part of the dystrophin-glycoprotein complex.

The α7β1 integrin is a major laminin receptor expressed in skeletalmuscle. The α7β integrin plays an important role in the development ofneuromuscular and myotendinous junctions. In the adult, the α7β1integrin is concentrated at junctional sites and found inextrajunctional regions where it mediates the adhesion of the musclefibers to the extracellular matrix. Mice that lack the α7 chain developmuscular dystrophy that affects the myotendinous junctions. The absenceof α7 integrin results in defective matrix deposition at themyotendinous junction. Loss of the α7 integrin in γ-sarcoglycan miceresults in severe muscle pathology. Absence of the α7 integrin in mdxmice also results in severe muscular dystrophy, confirming that the α7β1integrin serves as a major genetic modifier for Duchenne and othermuscular dystrophies.

Mutations in the α7 gene are responsible for muscular dystrophy inhumans. A screen of 117 muscle biopsies from patients with undefinedmuscle disease revealed 3 which lacked the α7 integrin chain and hadreduced levels of β1D integrin chain. These patients exhibit delayeddevelopmental milestones and impaired mobility consistent with the rolefor the α7β1 integrin in neuromuscular and myotendinous junctiondevelopment and function.

Several lines of evidence suggest the α7 integrin may be important formuscle regeneration. For example, during embryonic development, the α7β1integrin regulates myoblast migration to regions of myofiber formation.It has been found that MyoD (myogenic determination protein)transactivates α7 integrin gene expression in vitro, which wouldincrease α7 integrin levels in activated satellite cells. Human, mouseand rat myoblast cell lines derived from satellite cells express highlevels of α7 integrin. Elevated α7 integrin mRNA and protein aredetected in the skeletal muscle of 5 week old mdx mice, which correlateswith the period of maximum muscle degeneration and regeneration. Inaddition, the α7β1 integrin associates with muscle specific α1-integrinbinding protein (MIBP), which regulates laminin deposition in C2C12myoblasts. Laminin provides an environment that supports myoblastmigration and proliferation. Finally, enhanced expression of the α7integrin in dystrophic skeletal muscle results in increased numbers ofsatellite cells.

To date, many efforts to cure or ameliorate muscular dystrophy involveenhancing expression of various components of the costameric network.However, these approaches, while showing some promise in vitro or intransgenic animals, typically do not demonstrate effective results inhumans nor provide methods through which therapy could be accomplishedin humans. Such routes of therapy are notoriously difficult toimplement.

However, it is also well known that direct administration of proteins,particularly large proteins, is very difficult. For example, large size,high charge, short half life, poor stability, high immunogenicity, andpoor membrane permeability can limit the bioavailability of administeredproteins. In addition, depending on the route of administration, asubject's natural physiological processes can attack and degradeadministered proteins. For example, although laminin is known to play arole in the extracellular matrix, it is a particularly large(typically >600 kD), highly charged molecule and consequentlydifficulties in its administration to patients would likely have beenanticipated. Accordingly, efforts to date have focused on moresophisticated treatments, rather than direct administration oftherapeutic substances.

SUMMARY

In various embodiments, the present disclosure provides a method oftreating a subject with laminin or a composition that includes laminin.For example, some embodiments provide methods of improving muscularhealth, such as enhancing muscle regeneration, maintenance, or repair ina subject by administering to the subject an effective amount of lamininor a composition comprising laminin, including fragments, derivatives,or analogs thereof. In a specific example, the laminin is a completelaminin protein. In further examples, the laminin is selected fromlaminin-1, laminin-2, laminin-4, and combinations thereof. In furtherexamples, the laminin or laminin composition includes a substance atleast substantially homologous to laminin-1, laminin-2, or laminin-4. Inyet further implementations, the laminin or laminin compositioncomprises a polypeptide at least substantially homologous to the lamininα1 chain.

In additional examples, the laminin or laminin composition consists oflaminin-1, laminin-2, laminin-4, and combinations thereof. In furtherexamples, the laminin or laminin composition consists of a substance atleast substantially homologous to laminin-1, laminin-2, or laminin-4. Inyet further implementations, the laminin or laminin composition consistsof a polypeptide at least substantially homologous to the laminin α1chain. In a specific example, the laminin or laminin composition doesnot include a laminin fragment, such as including only a completelaminin protein.

In yet another example, the laminin or laminin composition consistsessentially of laminin-1, laminin-2, laminin-4, and combinationsthereof. In further examples, the laminin or laminin compositionconsists essentially of a substance at least substantially homologous tolaminin-1, laminin-2, or laminin-4. In yet further implementations, thelaminin or laminin composition consists essentially of a polypeptide atleast substantially homologous to the laminin α1 chain. In a specificexample, the laminin or laminin composition does not include a lamininfragment, such as including essentially only a complete laminin protein.

Further implementations of the disclosed method include diagnosing thesubject as having a condition treatable by administering laminin or acomposition comprising laminin. In one example, the subject is diagnosedas suffering from muscular dystrophy, such as a congenital musculardystrophy, Duchenne muscular dystrophy, or Limb-girdle musculardystrophy. In further instances the condition is characterized by thefailure of a subject, or the reduced ability of the subject, to expressone or more proteins associated with the formation or maintenance of theextracellular matrix, such as impaired or non-production of a laminin,an integrin, dystrophin, utrophin, or dystroglycan.

In a specific embodiment, the present disclosure also provides a methodfor increasing muscle regeneration in a subject. For example, geriatricsubjects, subjects suffering from muscle disorders, and subjectssuffering from muscle injury, including activity induced muscle injury,such as injury caused by exercise, may benefit from this embodiment.

In yet further embodiments of the disclosed method, the laminin orlaminin composition is administered in a preventative manner, such as toprevent or reduce muscular damage or injury (such as activity orexercise induced injury). For example, geriatric subjects, subjectsprone to muscle damage, or subjects at risk for muscular injury, such asathletes, may be treated in order to eliminate or ameliorate musculardamage, injury, or disease.

Implementations of the present disclosure may also be used to promotewound healing. In some examples, a laminin or a composition comprisinglaminin is administered into or proximate to a wound. In furtherexamples, the substance is administered systemically. Although thesubstance is typically applied after the wound occurs, the substance isapplied prospectively in some examples.

In further embodiments, the method of the present disclosure includesadministering the laminin or laminin composition with one or moreadditional pharmacological substances, such as a therapeutic agent. Insome aspects, the additional therapeutic agent enhances the therapeuticeffect of the laminin or laminin composition. In further aspects, thetherapeutic agent provides independent therapeutic benefit for thecondition being treated. In various examples, the additional therapeuticagent is a component of the extracellular matrix, such as an integrin,dystrophin, dystroglycan, utrophin, or a growth factor. In furtherexamples, the therapeutic agent reduces or enhances expression of asubstance that enhances the formation or maintenance of theextracellular matrix.

In some examples, the laminin or laminin composition is applied to aparticular area of the subject to be treated. For example, the lamininor laminin composition may be injected into a particular area to betreated, such as a muscle. In further examples, the laminin or laminincomposition is administered such that it is distributed to multipleareas of the subject, such as systemic administration or regionaladministration.

Laminin, or a composition comprising laminin, can be administered by anysuitable method, such as topically, parenterally (such as intravenouslyor intraperitoneally), or orally. In a specific example, the laminin orlaminin composition is administered systemically, such as throughparenteral administration, such as stomach injection or peritonealinjection.

Although the disclosed methods generally have been described withrespect to muscle regeneration, the disclosed methods also may be usedto enhance repair or maintenance, or prevent damage to, other tissuesand organs. For example, the methods of the present disclosure can beused to treat symptoms of muscular dystrophy stemming from effects tocells or tissue other than skeletal muscle, such as impaired or alteredbrain function, smooth muscles, or cardiac muscles.

There are additional features and advantages of the various embodimentsof the present disclosure. They will become evident from the followingdisclosure.

In this regard, it is to be understood that this is a brief summary ofthe various embodiments described herein. Any given embodiment of thepresent disclosure need not provide all features noted above, nor mustit solve all problems or address all issues in the prior art notedabove.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is immunofluorescence images of myofibers isolated fromnestin-GFP transgenic mice using an anti-α7 integrin monoclonalantibody.

FIG. 2 is photographs of the tibialis anterior muscle of wild-type andα7 integrin null mice 4, 10, and 28 days after cardiotoxin-inducedinjury.

FIG. 3 provides a graph illustrating Evan's blue dye uptake by wild-typeand α7 integrin null mice.

FIG. 4 is photomicrographs of hematoxylin and eosin stainings of tissuesections from wild-type and α7 integrin null mice.

FIG. 5 provides a graph illustrating the percentage of centrally locatednuclei in wild-type and α7 integrin null mice.

FIG. 6 provides a graph of embryonic myosin heavy chain expression inwild-type and α7 integrin null mice.

FIG. 7 provides a graph of myofiber cross-sectional area for wild-typeand α7 integrin null mice.

FIG. 8 provides a graph illustrating BrdU incorporation into wild-typeand α7 integrin null mice.

FIG. 9 provides a graph illustrating Pax7 expression in wild-type and α7integrin null mice.

FIG. 10 provides a graph illustrating MyoD expression in wild-type andα7 integrin null mice.

FIG. 11 is immunofluorescence images of myofibers isolated fromwild-type and α7 integrin null mice treated with laminin-1.

FIG. 12 is photographs of the tibialis anterior muscle of wild-type andα7 integrin null mice treated with laminin-14, 10, and 28 days aftercardiotoxin-induced injury.

FIG. 13 provides a graph illustrating Evan's blue dye update bywild-type and α7 integrin null mice treated with laminin-1.

FIG. 14 is photomicrographs of hematoxylin and eosin stainings of tissuesections from wild-type and α7 integrin null mice treated withlaminin-1.

FIG. 15 provides a graph illustrating the percentage of centrallylocated nuclei in wild-type and α7 integrin null mice treated withlaminin-1.

FIG. 16 provides a graph of embryonic myosin heavy chain expression inwild-type and α7 integrin null mice treated with laminin-1.

FIG. 17 provides a graph of myofiber cross-sectional area for wild-typeand α7 integrin null mice treated with laminin-1.

FIG. 18 provides a graph illustrating BrdU incorporation into wild-typeand α7 integrin null mice treated with laminin-1.

FIG. 19 provides a graph illustrating Pax7 expression in wild-type andα7 integrin null mice treated with integrin.

FIG. 20 provides a graph illustrating MyoD expression in wild-type andα7 integrin null mice treated with laminin.

FIG. 21 is an image of X-gal staining demonstrating that α7βgal^(+/−)myoblasts express β-galactosidase (left panel) which increases upondifferentiation to myotubes (right panel).

FIG. 22 is an image of a Western analysis of α7 integrin andβ-galactosidase expression in α7βgal^(+/−) cells differentiated from0-72 hours.

FIG. 23 is fluorescence-activated sorting (FACS) graphs (log of sidescatter versus FITC staining (intensity)) demonstrating thatα7βgal^(+/−) myoblasts exhibit increased β-galactosidase expressionfollowing 100 nM LAM-111 treatment.

FIG. 24 is an image of a Western analysis of α7B integrin and Cox-1expression in laminin-111- and phosphate-buffered saline-treated C2C12and Duchenne muscular dystrophy myoblasts.

FIG. 25 provides a graph (pixels versus square millimeters) of α7Bintegrin expression in laminin-111- and phosphate-bufferedsaline-treated C2C12 and Duchenne muscular dystrophy myoblasts.

FIG. 26 is immunofluorescence images (scale bar=10 μm) of the tibialisanterior muscle of control, phosphate-buffered saline-treated, andlaminin-111-treated muscle, illustrating the absence of dystrophin inmdx muscle treated with laminin-111 or phosphate-buffered saline andthat, while wild-type and phosphate-buffered saline-injected mdx musclelacked laminin-111, laminin-111 was detected in the extracellular matrixof laminin-111-injected mdx muscle.

FIG. 27 is photomicrographs (scale bar=10 μm) of hematoxylin and eosin(top panels) staining and Evans blue dye (EBD) uptake (bottom panels)for wild-type, phosphate-buffered saline-injected mdx muscle, andlaminin-111-injected mdx muscle, illustrating that laminin-111-injectedmuscle exhibited reduced centrally located nuclei and EBD uptakecompared to phosphate-buffered saline-injected mdx muscle.

FIG. 28 provides graphs of Evans blue dye uptake (left graph, percentageof positive fibers) and centrally located nuclei (right graph,percentage of fibers positive for centrally located nuclei) forwild-type, mdx muscle injected with phosphate-buffered saline, and mdxmuscle treated with laminin-111.

FIG. 29 is immunofluorescence images (scale bar=10 μm) of wild typemuscle, phosphate-buffered saline-treated mdx muscle, andlaminin-111-treated mdx muscle illustrating the presence or absence ofα7 integrin, utrophin, and α-bungarotoxin.

FIG. 30 is an image of a Western analysis of dystrophin, utrophin, α7Aintegrin, α7B integrin, β1D integrin, and Cox-1 expression in wild-typemuscle, phosphate-buffered saline-treated mdx muscle, andlaminin-111-treated mdx muscle.

FIG. 31 provides graphs of the ratio of α7A integrin/Cox-1 (top graph),α7B integrin/Cox-1 (middle graph), and utrophin/Cox-1 (bottom graph) forwild-type muscle, phosphate-buffered saline-treated mdx muscle, andlaminin-111-treated mdx muscle.

FIG. 32 is immunofluorescence images (scale bar=10 μm) of wild typemuscle, phosphate-buffered saline-treated mdx muscle, andlaminin-111-treated mdx muscle illustrating that a single 1 mg/kg doseof laminin-111 protein delivered intraperionteally in mdx mice resultedin localization to the heart, diaphragm and gastrocnemius.

FIG. 33 is immunofluorescence images of the diaphragm of mdx micetreated with phosphate-buffered saline (left images) or laminin-111(right images), illustrating that laminin-111 was located throughout thediaphragm of mdx mice following intraperitoneal injection withlaminin-111.

FIG. 34 provides graphs of creatine (mg/dl) and blood urea nitrogen(mg/dl) levels for wild-type muscle, mdx muscle injected withphosphate-buffered saline, and mdx muscle treated with laminin-111.

DETAILED DESCRIPTION Abbreviations

-   -   PBS—phosphate-buffered saline    -   LAM-111—laminin-1, which includes the chains α1β1γ1    -   NaCl—sodium chloride    -   NaOH—sodium hydroxide    -   HCl—hydrochloric acid    -   MCMD, MDC1A—merosin-deficient congenital muscular dystrophy    -   DMSO—dimethylsulfoxide    -   EDTA—ethylenediaminetetraacetic acid    -   eMyHC—embryonic myosin heavy chain    -   BrdU—bromodeoxyuridine    -   TA—tibialis anterior    -   H&E—hematoxylin and eosin    -   GFP—green fluorescent protein    -   WT—wild-type    -   EBD—Evan's blue dye    -   DMD—Duchenne muscular dystrophy    -   CLN—centrally located nuclei    -   nmol—nanomole    -   nM—nanomolar    -   MyoD—myogenic determination protein    -   MIBP—muscle specific α1-integrin binding protein    -   FACS—fluorescence activated sorting    -   FITC—fluorescein isothiocyanate    -   Pax7—paired box gene 7    -   Pax3—paired box gene 3    -   Cox-1—cyclooxygenase-1    -   MRF4—myogenic factor 6

Terms

In order to facilitate an understanding of the embodiments presented,the following explanations are provided.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. In case of conflict,the present specification, including explanations of terms, willcontrol. The singular terms “a,” “an,” and “the” include pluralreferents unless context clearly indicates otherwise. Similarly, theword “or” is intended to include “and” unless the context clearlyindicates otherwise. The term “comprising” means “including;” hence,“comprising A or B” means including A or B, or including A and B. Allnumerical ranges given herein include all values, including end points(unless specifically excluded) and any and all intermediate rangesbetween the endpoints.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present disclosure,suitable methods and materials are described herein. The disclosedmaterials, methods, and examples are illustrative only and not intendedto be limiting.

“Muscle” refers to any myoblast, myocyte, myofiber, myotube or otherstructure composed of muscle cells. Muscles or myocytes can be skeletal,smooth, or cardiac. Muscle may also refer to, in particularimplementations of the present disclosure, cells or other materialscapable of forming myocytes, such as stem cells and satellite cells.

“Extracellular matrix” refers to the extracellular structure of a tissueor a layer thereof, including the arrangement, composition, and forms ofone or more matrix components, such as proteins, including structuralproteins such as collagen and elastin, proteins such as fibronectin andlaminins, and proteoglycans. The matrix may comprise fibrillic collagen,having a network of fibers. In some examples, the extracellular matrixis connected to cells through the costameric protein network.

“Tissue” refers to an aggregate of cells, usually of a particular kind,together with their intercellular substance that form one of thestructural materials of an animal and that in animals include connectivetissue, epithelium, muscle tissue, and nerve tissue.

“Subject” refers to an organism, such as an animal, to whom treatmentsare administered. Subjects include mammals, such as humans, pigs, rats,cows, mice, dogs, cats, and primates.

“Laminin” refers to any of the family of glycoproteins that aretypically involved in the formation and maintenance of extracellularmatrices. Laminin is a heterotrimers formed from an α chain, a β chain,and a γ chain. The various chains of a particular laminin can affect theproperties of the molecule. In some aspects of the present disclosure,fragments, derivatives, or analogs of various laminins can be used, suchas laminins having at least a portion at least substantially homologousto the laminin α1 chain.

“At least substantially homologous,” as used in the present disclosure,refers to a degree of homology sufficient to produce at least a portionof the activity of a reference material in muscle regeneration,maintenance or repair, or wound healing. In some examples, materials areat least substantially homologous when they are at least about 95%, atleast about 98%, or at least about 99% homologous to a referencematerial.

A “fragment,” as used herein, refers to a portion of a substance, suchas laminin. A fragment may be, in some examples, a particular domain orchain of a protein. For example, particular embodiments of the presentdisclosure involve administering a fragment of laminin-1 correspondingto at least a portion of (or all of) the laminin α1 chain. Fragments maybe synthetic or may be derived from larger parent substances.

A “derivative,” as used herein, refers to a form of a substance, such asa laminin or portion thereof, which has at least one functional groupaltered, added, or removed, compared with the parent compound.

“Functional group” refers to a radical, other than a hydrocarbonradical, that adds a physical or chemical property to a substance.

As used herein, an “analog” refers to a compound which is sufficientlyhomologous to a compound such that it has a similar functional activityfor a desired purpose as the original compound. Analogs includepolypeptides having one or more amino acid substitutions compared with aparticular substance.

In some aspects, laminins may be administered as a mixture of laminins,including fragments, analogs, and derivatives thereof. Suitable methodsfor preparing analogs of laminin domains are disclosed in U.S. Pat. No.6,933,280, incorporated by reference herein to the extent notinconsistent with this disclosure.

The laminin materials or compositions of the present disclosure may bedelivered as discrete molecules or may be complexed with, or conjugatedto, another substance. For example, the laminin may be combined with acarrier, such as to aid in delivery of the laminin to a site of interestor to increase physiological uptake or incorporation of the laminin.

In specific examples, the laminin administered includes or consists oflaminin-1 (LAM-111), which includes the chains α1β1γ1. In furtherexamples, the laminin administered includes or consists of laminin-2,which includes the chains α2β1γ1. In yet further examples, the lamininadministered includes or consists of laminin-4, which includes thechains α2β2γ1.

Laminins may be obtained from any suitable source. For example,laminin-1 may be obtained from placental tissue or fromEngelbreth-Holm-Swarm murine sarcoma. Suitable methods of isolatingvarious laminins are disclosed in U.S. Pat. No. 5,444,158, incorporatedby reference herein to the extent not inconsistent with the presentdisclosure.

“Biological source” refers to an organism, such as an animal, such as amammal, or portion thereof, from which biological materials may beobtained. Examples of such materials include tissue samples, such asplacental material or sarcoma; cells, such as satellite cells;extracellular material, including laminins or other components thereof;or other organic or inorganic material found in the organism.

“Improving muscular health” refers to an improvement in muscular healthcompared with a preexisting state or compared with a state which wouldoccur in the absence of treatment. For example, improving muscularhealth may include enhancing muscle regeneration, maintenance, orrepair. Improving muscular health may also include prospectivelytreating a subject to prevent or reduce muscular damage or injury.

“Regeneration” refers to the repair of cells or tissue, such as musclecells or tissue (or organs) which includes muscle cells, followinginjury or damage to at least partially restore the muscle or tissue to acondition similar to which the cells or tissue existed before the injuryor damage occurred. Regeneration also refers to facilitating repair ofcells or tissue in a subject having a disease affecting such cells ortissue to eliminate or ameliorate the effects of the disease. In morespecific examples, regeneration places the cells or tissue in the samecondition or an improved physiological condition as before the injury ordamage occurred or the condition which would exist in the absence ofdisease.

“Maintenance” of cells or tissue, such as muscle cells or tissue (ororgans) which includes muscle cells, refers to maintaining the cells ortissue in at least substantially the same physiological condition, suchas maintaining such condition even in the presence of stimulus whichwould normally cause damage, injury, or disease.

“Repair” of cells or tissue, such as muscle cells or tissue (or organs)which includes muscle cells, refers to the physiological process ofhealing damage to the cells or tissue following damage or other trauma.

“Administering” refers to providing one or more substances to a subjectsuch that the subject may receive therapeutic benefit from thesubstance. The laminin, laminin composition, or other therapeuticsubstance are in general administered topically, nasally, intravenously,orally, intracranially, intramuscularly, parenterally or as implants,but even rectal or vaginal use is possible in principle. Laminin, orcompositions thereof, also may be administered to a subject using acombination of these techniques.

Suitable solid or liquid pharmaceutical preparation forms are, forexample, aerosols, (micro)capsules, creams, drops, drops or injectablesolution in ampoule form, emulsions, granules, powders, suppositories,suspensions, syrups, tablets, coated tablets, and also preparations withprotracted release of active compounds, in whose preparation excipientsand additives and/or auxiliaries such as binders, coating agents,disintegrants, flavorings, lubricants, solubilizers, sweeteners, orswelling agents are customarily used as described above. Thepharmaceutical compositions are suitable for use in a variety of drugdelivery systems. For a brief review of various methods for drugdelivery, see Langer, “New Methods of Drug Delivery,” Science249:1527-1533 (1990), incorporated by reference herein to the extent notinconsistent with the present disclosure.

The laminin, laminin compositions, or other therapeutic agents of thepresent disclosure can be formulated into therapeutically-activepharmaceutical compositions that can be administered to a subjectparenterally or orally. Parenteral administration routes include, butare not limited to epidermal, intraarterial, intramuscular (IM and depotIM), intraperitoneal (IP), intravenous (IV), intrasternal injection orinfusion techniques, intranasal (inhalation), intrathecal, injectioninto the stomach, subcutaneous injections (subcutaneous (SQ and depotSQ), transdermal, topical, and ophthalmic.

The laminin, laminin composition, or other therapeutic agent can bemixed or combined with a suitable pharmaceutically acceptable excipientsto prepare pharmaceutical compositions. Pharmaceutically acceptableexcipients include, but are not limited to, alumina, aluminum stearate,buffers (such as phosphates), glycine, ion exchangers (such as to helpcontrol release of charged substances), lecithin, partial glyceridemixtures of saturated vegetable fatty acids, potassium sorbate, serumproteins (such as human serum albumin), sorbic acid, water, salts orelectrolytes such as cellulose-based substances, colloidal silica,disodium hydrogen phosphate, magnesium trisilicate, polyacrylates,polyalkylene glycols, such as polyethylene glycol,polyethylene-polyoxypropylene-block polymers, polyvinyl pyrrolidone,potassium hydrogen phosphate, protamine sulfate, group 1 halide saltssuch as sodium chloride, sodium carboxymethylcellulose, waxes, wool fat,and zinc salts, for example. Liposomal suspensions may also be suitableas pharmaceutically acceptable carriers.

Upon mixing or addition of the laminin, laminin composition, or othertherapeutic agent, the resulting mixture may be a solid, solution,suspension, emulsion, or the like. These may be prepared according tomethods known to those of ordinary skill in the art. The form of theresulting mixture depends upon a number of factors, including theintended mode of administration and the solubility of the agent in theselected carrier.

Pharmaceutical carriers suitable for administration of the laminin,laminin composition, or other therapeutic agent include any suchcarriers known to be suitable for the particular mode of administration.In addition, the laminin, laminin composition, or other therapeuticsubstance can also be mixed with other inactive or active materials thatdo not impair the desired action, or with materials that supplement thedesired action, or have another action.

Methods for solubilizing may be used where the agents exhibitinsufficient solubility in a carrier. Such methods are known andinclude, but are not limited to, dissolution in aqueous sodiumbicarbonate, using cosolvents such as dimethylsulfoxide (DMSO), andusing surfactants such as TWEEN® (ICI Americas, Inc., Wilmington, Del.).

The laminin, laminin composition, or other therapeutic agent can beprepared with carriers that protect them against rapid elimination fromthe body, such as coatings or time-release formulations. Such carriersinclude controlled release formulations, such as, but not limited to,microencapsulated delivery systems. The laminin, laminin composition, orother therapeutic agent is included in the pharmaceutically acceptablecarrier in an amount sufficient to exert a therapeutically usefuleffect, typically in an amount to avoid undesired side effects, on thetreated subject. The therapeutically effective concentration may bedetermined empirically by testing the compounds in known in vitro and invivo model systems for the treated condition. For example, mouse modelsof muscular dystrophy may be used to determine effective amounts orconcentrations that can then be translated to other subjects, such ashumans, as known in the art.

Injectable solutions or suspensions can be formulated, using suitablenon-toxic, parenterally-acceptable diluents or solvents, such as1,3-butanediol, isotonic sodium chloride solution, mannitol, Ringer'ssolution, saline solution, or water; or suitable dispersing or wettingand suspending agents, such as sterile, bland, fixed oils, includingsynthetic mono- or diglycerides, and fatty acids, including oleic acid;a naturally occurring vegetable oil such as coconut oil, cottonseed oil,peanut oil, sesame oil, and the like; glycerine; polyethylene glycol;propylene glycol; or other synthetic solvent; antimicrobial agents suchas benzyl alcohol and methyl parabens; antioxidants such as ascorbicacid and sodium bisulfite; buffers such as acetates, citrates, andphosphates; chelating agents such as ethylenediaminetetraacetic acid(EDTA); agents for the adjustment of tonicity such as sodium chlorideand dextrose; and combinations thereof. Parenteral preparations can beenclosed in ampoules, disposable syringes, or multiple dose vials madeof glass, plastic, or other suitable material. Buffers, preservatives,antioxidants, and the like can be incorporated as required. Whereadministered intravenously, suitable carriers include physiologicalsaline, phosphate-buffered saline (PBS), and solutions containingthickening and solubilizing agents such as glucose, polyethylene glycol,polypropyleneglycol, and mixtures thereof. Liposomal suspensions,including tissue-targeted liposomes, may also be suitable aspharmaceutically acceptable carriers.

For topical application, the laminin, laminin composition, or othertherapeutic agent may be made up into a cream, lotion, ointment,solution, or suspension in a suitable aqueous or non-aqueous carrier.Topical application can also be accomplished by transdermal patches orbandages which include the therapeutic substance. Additives can also beincluded, e.g., buffers such as sodium metabisulphite or disodiumedetate; preservatives such as bactericidal and fungicidal agents,including phenyl mercuric acetate or nitrate, benzalkonium chloride, orchlorhexidine; and thickening agents, such as hypromellose.

If the laminin, laminin composition, or other therapeutic agent isadministered orally as a suspension, the pharmaceutical compositions canbe prepared according to techniques well known in the art ofpharmaceutical formulation and may contain a suspending agent, such asalginic acid or sodium alginate, bulking agent, such as microcrystallinecellulose, a viscosity enhancer, such as methylcellulose, andsweeteners/flavoring agents. Oral liquid preparations can containconventional additives such as suspending agents, e.g., gelatin, glucosesyrup, hydrogenated edible fats, methyl cellulose, sorbitol, and syrup;emulsifying agents, e.g., acacia, lecithin, or sorbitan monooleate;non-aqueous carriers (including edible oils), e.g., almond oil,fractionated coconut oil, oily esters such as glycerine, propyleneglycol, or ethyl alcohol; preservatives such as methyl or propylp-hydroxybenzoate or sorbic acid; and, if desired, conventionalflavoring or coloring agents. When formulated as immediate releasetablets, these compositions can contain dicalcium phosphate, lactose,magnesium stearate, microcrystalline cellulose, and starch and/or otherbinders, diluents, disintegrants, excipients, extenders, and lubricants.

If oral administration is desired, the laminin, laminin composition, orother therapeutic substance can be provided in a composition thatprotects it from the acidic environment of the stomach. For example, thelaminin, laminin composition, or other therapeutic agent can beformulated with an enteric coating that maintains its integrity in thestomach and releases the active compound in the intestine. The laminin,laminin composition, or other therapeutic agent can also be formulatedin combination with an antacid or other such ingredient.

Oral compositions generally include an inert diluent or an ediblecarrier and can be compressed into tablets or enclosed in gelatincapsules. For the purpose of oral therapeutic administration, thelaminin, laminin composition, or other therapeutic substance can beincorporated with excipients and used in the form of capsules, tablets,or troches. Pharmaceutically compatible adjuvant materials or bindingagents can be included as part of the composition.

The capsules, pills, tablets, troches, and the like can contain any ofthe following ingredients or compounds of a similar nature: a bindersuch as, but not limited to, acacia, corn starch, gelatin, gumtragacanth, polyvinylpyrrolidone, or sorbitol; a filler such as calciumphosphate, glycine, lactose, microcrystalline cellulose, or starch; adisintegrating agent such as, but not limited to, alginic acid and cornstarch; a lubricant such as, but not limited to, magnesium stearate,polyethylene glycol, silica, or talc; a gildant, such as, but notlimited to, colloidal silicon dioxide; a sweetening agent such assucrose or saccharin; disintegrants such as potato starch; dispersing orwetting agents such as sodium lauryl sulfate; and a flavoring agent suchas peppermint, methyl salicylate, or fruit flavoring.

When the dosage unit form is a capsule, it can contain, in addition tomaterial of the above type, a liquid carrier, such as a fatty oil. Inaddition, dosage unit forms can contain various other materials thatmodify the physical form of the dosage unit, for example, coatings ofsugar and other enteric agents. The laminin, laminin composition, orother therapeutic agent can also be administered as a component of anelixir, suspension, syrup, wafer, tea, chewing gum, or the like. A syrupmay contain, in addition to the active compounds, sucrose or glycerin asa sweetening agent and certain preservatives, dyes and colorings, andflavors.

When administered orally, the compounds can be administered in usualdosage forms for oral administration. These dosage forms include theusual solid unit dosage forms of tablets and capsules as well as liquiddosage forms such as solutions, suspensions, and elixirs. When the soliddosage forms are used, they can be of the sustained release type so thatthe compounds need to be administered less frequently.

As explained elsewhere in the present disclosure, surprisingly andcontrary to prior expectations, it has been determined that laminin isreadily absorbed by subjects and made physiologically available. Forexample, it has been demonstrated that laminin injected into the stomachof a subject is incorporated systemically in the subject, such as indiverse muscle groups. Intraperitoneal injection also produced systemicdistribution of laminin, including distribution of laminin to thediaphragm, gastrocnemius muscles, and cardiac muscles. In furtherexamples, when administration occurs by intramuscular injection, thelaminin has been found to permeate to nearby muscle groups. Accordingly,it is believed the administration of laminin may not suffer from some ofthe severe delivery problems which have plagued other proteins,particularly large proteins. Examples of methods and compositions foradministering therapeutic substances which include proteins includethose discussed in Banga, Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems 2ed. (2005); Mahato,Biomaterials for Delivery and Targeting of Proteins and Nucleic Acids(2004); McNally, Protein Formulation and Delivery, 2ed. (2007); andKumar et al., “Novel Delivery Technologies for Protein and PeptideTherapeutics,” Current Pharm. Biotech., 7:261-276 (2006); each of whichis incorporated by reference herein to the extent not inconsistent withthe present disclosure.

“Inhibiting” a disease or condition refers to inhibiting the developmentof a disease or condition, for example, in a subject who is at risk fora disease or who has a particular disease. Particular methods of thepresent disclosure provide methods for inhibiting muscular dystrophy.“Treatment” refers to a therapeutic intervention that ameliorates a signor symptom of a disease or condition after it has begun to develop. Asused herein, the term “ameliorating,” with reference to a disease orcondition, refers to any observable beneficial effect of the treatment.The beneficial effect can be evidenced, for example, by a delayed onsetof clinical symptoms of the disease or condition in a susceptiblesubject, a reduction in severity of some or all clinical symptoms of thedisease or condition, a slower progression of the disease or condition,a reduction in the number of relapses of the disease or condition, animprovement in the overall health or well-being of the subject, by otherparameters well known in the art that are specific to the particulardisease or condition, and combinations of such factors.

“Therapeutically-effective amount” refers to an amount effective forlessening, ameliorating, eliminating, preventing, or inhibiting at leastone symptom of a disease, disorder, or condition treated and may beempirically determined. In various embodiments of the presentdisclosure, a “therapeutically-effective amount” is a “muscleregeneration promoting-amount,” an amount sufficient to achieve astatistically significant promotion of tissue or cell regeneration, suchas muscle cell regeneration, compared to a control.

In particular, indicators of muscular health, such as muscle cellregeneration, maintenance, or repair, can be assessed through variousmeans, including monitoring markers of muscle regeneration, such astranscription factors such as Pax7, Pax3, MyoD, MRF4, and myogenin. Forexample, increased expression of such markers can indicate that muscleregeneration is occurring or has recently occurred. Markers of muscleregeneration, such as expression of embryonic myosin heavy chain(eMyHC), can also be used to gauge the extent of muscle regeneration,maintenance, or repair. For example, the presence of eMyHC can indicatethat muscle regeneration has recently occurred in a subject.

Muscle cell regeneration, maintenance, or repair can also be monitoredby determining the girth, or mean cross sectional area, of muscle cellsor density of muscle fibers. Additional indicators of muscle conditioninclude muscle weight and muscle protein content. Mitotic index (such asby measuring BrdU incorporation) and myogenesis can also be used toevaluate the extent of muscle regeneration.

In particular examples, the improvement in muscle condition, such asregeneration, compared with a control is at least about 10%, such as atleast about 30%, or at least about 50% or more.

In some implementations, the effective amount of laminin or laminincomposition is administered as a single dose per time period, such asevery three or four months, month, week, or day, or it can be dividedinto at least two unit dosages for administration over a period.Treatment may be continued as long as necessary to achieve the desiredresults. For instance, treatment may continue for about 3 or 4 weeks upto about 12-24 months or longer, including ongoing treatment. Thecompound can also be administered in several doses intermittently, suchas every few days (for example, at least about every two, three, four,five, or ten days) or every few weeks (for example at least about everytwo, three, four, five, or ten weeks).

Particular dosage regimens can be tailored to a particular subject,condition to be treated, or desired result. For example, when themethods of the present disclosure are used to treat muscular dystrophyor similar conditions, an initial treatment regimen can be applied toarrest the condition. Such initial treatment regimen may includeadministering a higher dosage of the laminin or laminin composition, oradministering such material more frequently, such as daily. After adesired therapeutic result has been obtained, such as a desired level ofmuscle regeneration, a second treatment regimen may be applied, such asadministering a lower dosage or laminin or laminin composition oradministering such material less frequently, such as monthly,bi-monthly, quarterly, or semi-annually. In such cases, the secondregimen may serve as a “booster” to restore or maintain a desired levelof muscle regeneration. Similar treatment regimens may be used for othersubjects with reduced or impaired muscle regeneration capabilities, suchas geriatric subjects.

When particular methods of the present disclosure are used to prevent ormitigate muscle damage, such as damage caused by exertion or injury, thesubject is typically treated a sufficient period of time before theexertion or injury in order to provide therapeutic effect. For example,the subject may be treated at least about 24 hours before the expectedactivity or potential injury, such as at least about 48 hours, about 72hours, about 1 week, about 2 weeks, about three weeks, or about 4 weeksor more prior.

When embodiments of the method of the present disclosure are used topromote wound healing, the laminin, laminin composition, or othertherapeutic substance can be applied directly to, or proximately to, thearea to be treated. For example, the substance can be injected into ornear the area. In further examples, the substance can be appliedtopically to the area to be treated. Treatment is typically initiatedprior to the injury to several weeks following the injury. In morespecific implementations, the treatment is initiated between about 12and about 72 hours following injury, such as between about 24 and about48 hours following injury. In some cases, a single administration of thesubstance is effective to provide the desired therapeutic effect. Infurther examples, additional administrations are provided in order toachieve the desired therapeutic effect.

Amounts effective for various therapeutic treatments of the presentdisclosure may, of course, depend on the severity of the disease and theweight and general state of the subject, as well as the absorption,inactivation, and excretion rates of the therapeutically-active compoundor component, the dosage schedule, and amount administered, as well asother factors known to those of ordinary skill in the art. It alsoshould be apparent to one of ordinary skill in the art that the exactdosage and frequency of administration will depend on the particularlaminin, laminin composition, or other therapeutic substance beingadministered, the particular condition being treated, the severity ofthe condition being treated, the age, weight, general physical conditionof the particular subject, and other medication the subject may betaking. Typically, dosages used in vitro may provide useful guidance inthe amounts useful for in vivo administration of the pharmaceuticalcomposition, and animal models may be used to determine effectivedosages for treatment of particular disorders. For example, mouse modelsof muscular dystrophy may be used to determine effective dosages thatcan then be translated to dosage amount for other subjects, such ashumans, as known in the art. Various considerations in dosagedetermination are described, e.g., in Gilman et al., eds., Goodman AndGilman's: The Pharmacological Bases of Therapeutics, 8th ed., PergamonPress (1990); and Remington's Pharmaceutical Sciences, 17th ed., MackPublishing Co., Easton, Pa. (1990), each of which is herein incorporatedby reference to the extent not inconsistent with the present disclosure.

In specific examples, the laminin or laminin composition is administeredto a subject in an amount sufficient to provide a dose of laminin ofbetween about 10 fmol/g and about 500 nmol/g, such as between about 2nmol/g and about 20 nmol/g or between about 2 nmol/g and about 10nmol/g. In additional examples, the laminin or laminin composition isadministered to a subject in an amount sufficient to provide a dose oflaminin of between about 0.01 μg/kg and about 1000 mg/kg or betweenabout 0.1 mg/kg and about 1000 mg/kg, in particular examples this amountis provided per day or per week. In another example, the laminin orlaminin composition is administered to a subject in an amount sufficientto provide a dose of laminin of between about 0.2 mg/kg and about 2mg/kg. In further examples, the laminin or laminin composition isadministered to a subject in an amount sufficient to provide aconcentration of laminin in the administrated material of between about5 nM and about 500 nM, such as between about 50 nM and about 200 nm, orabout 100 nM.

The above term descriptions are provided solely to aid the reader, andshould not be construed to have a scope less than that understood by aperson of ordinary skill in the art or as limiting the scope of theappended claims.

DESCRIPTION

Generally, the present disclosure provides embodiments of a method andcomposition for enhancing cell or tissue repair, regeneration, ormaintenance, including prospective treatment against subsequent injury,damage, or disease. In various embodiments, the present disclosureprovides methods of treating muscular dystrophy, enhancing muscle repairfollowing injury or damage, or reducing the severity of muscle injury ordamage. Further embodiments provide a method for enhancing woundhealing.

In some embodiments, the method includes administering an effectiveamount of laminin or a composition which includes an effective amount oflaminin. In a specific implementation of the method, the laminin islamanin-1. In a further specific implementation of the method, thelaminin is laminin-2 or laminin-4.

Without intending to be limited to a particular mechanism of action,laminin is believed to aid muscle regeneration by activating satellitecells to proliferate and differentiate into new muscle cells andmyotubes. Accordingly, muscle repair may be enhanced compared with thesubject's native condition.

Particularly when the methods are used to treat muscular dystrophy, andagain without being bound by a theory of operation, laminin may also aidin binding components of the extracellular matrix, such as binding todystrophin or α7β1 integrin. For example, in Duchenne musculardystrophy, increased amounts of laminin may aid in forming connectionswith the basement membrane through binding of α7β1 integrin or anotherreceptor, such as utrophin, which is homologous to dystrophin.Administration of laminin may also upregulate expression of one or morecomponents of the costameric network, such as utrophin or α7β1 integrin,potentially providing additional linkage points between theextracellular matrix and the remainder of the costamere. Laminin mayalso provide a structural environment to improve tissue integrity.

In further embodiments, the present disclosure provides methods forpromoting muscle regeneration. Muscle regeneration may benefit, forexample, geriatric or other patient populations with reduced musclerepair capability, or simply speed the muscle repair process forotherwise physiologically unimpaired patients. In particularimplementations, administration of laminin can aid muscle repair, orreduction of muscle damage, in athletes or others havingactivity-induced muscle injury or damage. In yet furtherimplementations, muscle repair in patients suffering from muscle damage,such as through accident or injury, can be augmented by administrationof laminin.

In various examples of the embodiments of the present disclosure, thelaminin or laminin composition is administered with one or more othercomponents, such as components of the extracellular matrix. For example,the additional substance can include aggrecan, angiostatin, cadherins,collagens (including collagen I, collagen III, or collagen IV), decorin,elastin, enactin, endostatin, fibrin, fibronectin, osteopontin,tenascin, thrombospondin, vitronectin, and combinations thereof.Biglycans, glycosaminoglycans (such as heparin), glycoproteins (such asdystroglycan), proteoglycans (such as heparan sulfate), and combinationsthereof can also be administered. A particular laminin can beadministered with other forms of laminin, laminin analogs, lamininderivatives, or a fragment of any of the foregoing.

Growth stimulants may be added in conjunction with the laminin orlaminin composition. Examples of growth stimulants include cytokines,polypeptides, and growth factors such as brain-derived neurotrophicfactor (BDNF), CNF (ciliary neurotrophic factor), EGF (epidermal growthfactor), FGF (fibroblast growth factor), glial growth factor (GGF),glial maturation factor (GMF) glial-derived neurotrophic factor (GDNF),hepatocyte growth factor (HGF), insulin, insulin-like growth factors,kerotinocyte growth factor (KGF), nerve growth factor (NGF),neurotropin-3 and -4, PDGF (platelet-derived growth factor), vascularendothelial growth factor (VEGF), and combinations thereof.

Additional therapeutic agents can be added to enhance the therapeuticeffect of the laminin or laminin composition. For example, a source ofmuscle cells can be added to aid in muscle regeneration and repair. Insome aspects of the present disclosure, satellite cells are administeredto a subject in combination with laminin therapy. U.S. PatentPublication 2006/0014287, incorporated by reference herein to the extentnot inconsistent with the present disclosure, provides methods ofenriching a collection of cells in myogenic cells and administeringthose cells to a subject.

In further aspects, stem cells, such as adipose-derived stem cells, areadministered to the subject. Suitable methods of preparing andadministering adipose-derived stem cells are disclosed in U.S. PatentPublication 2007/0025972, incorporated by reference herein to the extentnot inconsistent with the present disclosure. Additional cellularmaterials, such as fibroblasts, can also be administered, in someexamples.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the invention to the particular features or embodiments described.

Example 1 Materials and Methods

Animals

Wild-type (C57BL/6), α7 integrin-null (C57BL/6 background), andNestin-GFP mouse (C57BL/6 background) used in these studies wereeuthanized in accordance with protocols approved by the University ofNevada, Reno and University of Washington, Seattle Institutional AnimalCare and Use Committees.

Histology

Tibialis anterior (TA) muscles were embedded in Optimal CuttingTemperature (OCT) (Tissue-Tek; Sakura Finetek, Torrance, Calif., UnitedStates) and 10 μm cryosections were cut (≧50 μm apart) using a Leica CM1850 cryostat placed on Surgipath microscope slides (Surgipath MedicalIndustries, Richmond, Ill.). Tissue sections were stained usinghematoxylin and eosin (H&E) as previously described in Rooney et al.,“Severe muscular dystrophy in mice that lack dystrophin and alphα7integrin,” J. Cell Sci. 119:2185-2195 (2006), incorporated by referenceherein to the extent not inconsistent with the present disclosure.Central myonuclei in regenerating muscles were counted at 630×magnification by bright-field microscopy. The number of central nucleiper muscle fiber was determined by counting a minimum of 1000 musclefibers per animal. At least five animals from each genotype wereanalyzed. In addition, the cross-sectional area was examined in aminimum of 5000 muscle fibers per group per time point. Results werereported as the average fiber cross-sectional area.

Immunofluorescence

TA muscles were embedded in Tissue-TEK Optimal Cutting Temperaturecompound (Sakura Finetek USA Inc., Torrance, Calif.). Sections were cutat 10 μm using a Leica CM1850 cryostat and placed onto Surgipathmicroscope slides (Surgipath Medical Industries, Richmond, Ill.).Laminin-α2 chain was detected with a 1:500 dilution of rabbitanti-laminin-α2 (2G) polyclonal antibody (a kind gift from PeterYurchenco, Robert Wood Johnson Medical School, Department of Pathology,Piscataway, N.J.). The laminin-al chain was detected using ananti-laminin-al antibody (sc-5582, Santa Cruz Biotechnology, Santa Cruz,Calif.). Primary rabbit antibodies were detected with a 1:500 dilutionof fluorescein isothiocyanate (FITC)-conjugated anti-rabbit secondaryantibody.

For mouse monoclonal antibodies, endogenous mouse immunoglobulin wasblocked with a mouse-on-mouse (MOM) kit (Vector Laboratories,Burlingame, Calif.). Expression of MyoD and Pax7 was detected using 5μg/ml anti-MyoD and anti-Pax7 (Developmental Studies Hybridoma Bank(DSHB), Iowa City, Iowa). eMyHC was detected as previously described(Rooney et al., 2006). A 1 μg/ml concentration oftetramethylrhodamine-conjugated wheat-germ agglutinin (WGA) (MolecularProbes, Eugene, Oreg.) was used to define muscle fibers. Fluorescencewas observed with a Zeiss Axioskop 2 Plus fluorescent microscope andimages were captured with a Zeiss AxioCam HRc digital camera andAxiovision 4.1 software (all available from Carl Zeiss Microlmaging,Thornwood, N.Y.). Multiple adjacent sections were analyzed within 20random, non-overlapping microscopic fields per animal at 630×magnification.

Single myofibers were isolated from the Extensor Digitorum Longus muscleof 10 week old nestin-GFP transgenic mice after collagenase digestionand cultured individually in Matrigel-coated wells as previouslydescribed (Shefer, et al., “Skeletal muscle satellite cells canspontaneously enter an alternative mesenchymal pathway,” J. Cell Sci.117:5393-5404 (2004); Shefer, et al., “Isolation and culture of skeletalmuscle myofibers as a means to analyze satellite cells,” Methods Mol.Biol. 290:281-304 (2005); each of which is incorporated by referenceherein to the extent not inconsistent with the present disclosure).Adherent single myofibers were fixed in 4% paraformaldehyde andincubated with 1:1000 dilution of anti-α7 integrin rat monoclonalantibody (CA5.5) (Sierra BioSource, Morgan Hill, Calif.). The anti-α7integrin rat antibody was detected using rhodamine labeled anti-ratsecondary antibody. Both GFP and rhodamine fluorescence were detectedusing an inverted fluorescent microscope (Nikon eclipse, TE2000-S, NikonInstruments, Inc., Melville, N.Y.) and images were acquired with aCoolSNAP_(ES) monochrome CCD camera (Princeton Instruments Inc.,Trenton, N.J.) controlled by MetaVue Imaging System (Universal ImagingCorporation, Downingtown, Pa.).

Evan's Blue Dye Assay

Mice were injected intraperitoneally with 50 μl of a 10 mg/ml solutionof sterile Evans blue dye (EBD) solution per 10 g of body weight. After3 hours, the TA muscle was harvested and flash-frozen in liquidnitrogen. 10 μm cryosections were placed on microscope slides and fixedin 4% paraformaldehyde. Muscle fibers were outlined by incubating tissuesections with Oregon Green-488-conjugated wheat germ agglutinin (2μg/ml, Molecular Probes, Eugene, Oreg.). A minimum of 1000 fibers peranimal were counted to determine the percentage of muscle fiberspositive for EBD. At least four animals from each genotype wereanalyzed. Images were captured and counting conducted at 630×magnification.

Bromodeoxyuridine (BrdU) Incorporation

BrdU (500 mg/kg) was injected intraperitoneally at 72 hours, 48 hoursand 24 hours prior to muscle harvesting. Muscle cryosections were fixedin 95% ethanol for 1 minute. Sections were then rinsed inphosphate-buffered saline (PBS) and treated with 2N hydrochloric acid(HCl) for 20 minutes. The sections were neutralized in 50 mM sodiumchloride (NaCl) for 20 minutes, followed by incubation in 100 mMTris-HCl for 20 minutes and rinsed in PBS. Tissue was incubated inanti-BrdU antibody (G3G4, 1:1000, Developmental Studies Hybridoma Bank(DSHB), Iowa City, Iowa) for 1 hour, washed in PBS and mounted inVectashield (Vector Labs, Burlingame, Calif.).

Cardiotoxin-Induced Muscle Injury

Mice were anesthetized with avertin (0.25 μl/g of body weight) and 100μl of a 10 μm cardiotoxin solution (C3987, Sigma, St. Louis, Mo.) in PBSwas injected into the left TA muscle of 5-week-old male wild-type andα7^(−/−) mice. The right TA muscles were injected with 100 μl of PBS andused as a control. The mice were euthanized and muscles harvested at 4,7, 10, and 28 days after cardiotoxin injection for analysis.

Laminin-111 Injections

Natural mouse laminin (Invitrogen, Carlsbad, Calif.) at 100 nM in PBSwas injected into the left TA muscle of anesthetized wild-type andα7^(−/−) mice three days prior to cardiotoxin injection. The right TAmuscles were injected with 100 μl of PBS and served as controls. Themuscles were harvested at 0, 4, 7, 10 and 28 days post-cardiotoxininjection for analysis.

Statistical Analysis

All averaged data are reported as the mean±standard deviation.Comparisons between multiple groups were performed by one-way-analysisof variance (ANOVA) for parametric data or by Kruskal-Wallisone-way-analysis of variance on ranks for non-parametric data usingSigmaStat 1.0 software (Jandel Corporation, San Rafael, Calif.). P<0.05was considered statistically significant.

Integrin Expression in Quiescent Satellite Cells

To confirm that the α7 integrin is expressed in satellite cells,isolated myofibers from nestin-GFP transgenic mice were subjected toimmunofluorescence using an anti-α7 integrin monoclonal antibody (FIG.1). Nestin-GFP is specifically expressed in quiescent satellite cells.All nestin-GFP positive cells on the myofiber surface were also positivefor the α7 integrin (FIG. 1). Image analysis indicated strongerlocalization of the α7 integrin on the basal surface of the satellitecells. These data confirm that quiescent satellite cells express the α7integrin and localization is enriched on the basal surface facing themuscle myofiber.

Muscle Repair in Integrin Null Mice

Recent studies suggest the α7 integrin plays a role in satellite cellactivation and/or proliferation. To examine if the α7β1 integrin isrequired for muscle repair, the tibialis anterior (TA) muscle ofwild-type and α7 integrin null mice were subjected tocardiotoxin-induced injury and examined 4, 10 and 28 days later (FIG.2). Four days after cardiotoxin injury, wild-type TA muscle appearedhealthy and this appearance persisted for 28 days. In contrast, α7integrin null muscle exhibited large white regions of damaged muscle at4 and 10 days post-injury. At 28 days, regions of muscle damage werestill clearly evident in α7 integrin null muscle. These data suggestloss of the α7 integrin in skeletal muscle results in a profound delayin muscle regeneration.

Loss of the α7 Integrin Results in Decreased Membrane Integrity afterInjury

To examine membrane integrity after cardiotoxin treatment, wild-type andα7 integrin null mice were injected with Evan's blue dye (EBD). EBDuptake was absent in both groups prior to cardiotoxin injection (FIG.3). Although α7 integrin null muscle was negative for EBD uptake priorto cardiotoxin injection, 7-fold more myofibers were EBD positive at day4 compared to wild-type. At day 4 post-injury, 8.5% of wild-type and 66%of α7 integrin null myofibers were EBD positive. After 10 days, lessthan 4% of wild-type myofibers were positive for EBD uptake, while 40%of α7 integrin deficient myofibers were EBD positive. At 28 dayspost-cardiotoxin injection, 17% of α7 integrin null muscle fibers werestill EBD positive, while EBD was not observed in wild-type muscle(P<0.05), (FIG. 3). These results indicate loss of the α7 integrinresults in increased sarcolemmal fragility after cardiotoxin-inducedinjury.

Reduced Muscle Repair in α7 Integrin Null Mice

Hematoxylin and Eosin (H&E) staining was used to examine mononuclearcell infiltrate and centrally located nuclei after cardiotoxin-inducedinjury (FIG. 4, scale bar indicates 10 μm). Four days aftercardiotoxin-injury, wild-type muscle exhibited mononuclear cellinfiltrate and myofibers containing centrally located nuclei (FIG. 4).By day 10, wild-type muscle exhibited little mononuclear infiltrate andmost myofibers contained centrally located nuclei. By 28 days inwild-type muscle, repair was complete and most myofibers containedcentrally located nuclei and little mononuclear cell infiltrate wasevident. In contrast, after 4 days post-cardiotoxin-induced damage, α7integrin null muscle exhibited extensive mononuclear cell infiltrate andhypotrophic muscle fibers which extended to 10 days post-cardiotoxininjury (FIG. 4). By 28 days, α7 integrin null muscle exhibitedhypotrophic myofibers which contained centrally located nuclei andmononuclear cell infiltrate.

To quantify muscle repair, the percentage of myofibers with centrallylocated nuclei was calculated (FIG. 5). In wild-type mice, 81.8% ofmuscle fibers contained centrally located nuclei 4 days post-cardiotoxininjury. In contrast only 28.1% of muscle fibers in α7 integrin nullmuscle were positive for centrally located nuclei (P<0.05). By 10 and 28days, 95.5% and 97.5% of muscle fibers, respectively, in wild-type miceexhibited centrally located nuclei. By days 10 and 28, 82% and 95.5% ofmuscle fibers in α7 integrin null muscle, respectively, exhibitedcentrally located nuclei which were lower than wild-type (P<0.05). Theseresults indicate loss of the α7 integrin results in delayed muscleregeneration.

Embryonic myosin heavy chain (eMyHC) is transiently expressed aftermuscle repair and used as a marker for recent muscle regeneration. Atday 0 there was an absence of eMyHC in both wild-type and α7 integrinnull mice (FIG. 6). At 4 and 10 days post-cardiotoxin treatment,expression of eMyHC was detected in over 99% of wild-type muscle fibers(FIG. 6). In sharp contrast, only 2.2% and 9.9% of α7 integrin nullmuscle fibers expressed eMyHC at 4 and 10 days respectively aftercardiotoxin-injury (FIG. 6). By day 28, only 11.3% of α7 integrin nullmyofibers were eMyHC positive, while 18.5% of wild-type muscle was eMyHCpositive (*P<0.001). These results confirm that loss of the α7 integrinresults in defective muscle repair as measured by the transientexpression of eMyHC.

Cardiotoxin Injury Results in Hypotrophic Muscle Fibers in α7 IntegrinNull Mice

To determine if loss of the α7 integrin affected muscle repair afterinjury, myofiber cross-sectional area was measured (FIG. 7).Regenerating muscle fibers in wild-type mice were 31% larger than α7integrin null muscle fibers 4 days post-cardiotoxin injury (FIG. 7). Atday 10, regenerating wild-type myofibers were 45.1% larger compared toα7 integrin-deficient muscle fibers (FIG. 7). By day 28, wild-typemuscle displayed muscle fiber size variation. However this was incontrast to the α7 integrin null muscle which continued to display smallcross-sectional areas, with the vast majority of fibers in the 100-600μm² range. These results indicate loss of the α7 integrin results inreduced regenerative capacity, giving rise to hypotrophic muscle fibers.

Satellite Cell Proliferation and Differentiation are Reduced in α7Integrin-Deficient Muscle after Injury

To determine if satellite cell proliferation was decreased in α7integrin null mice, incorporation of BrdU into the nuclei of satellitecells was quantified (FIG. 8). At 4 days post-injury, α7 integrin nullmuscle contained 3-fold fewer BrdU-positive nuclei compared to wild-typeanimals (FIG. 8). However, BrdU positive nuclei were increased in α7integrin null mice at days 10 and 28 compare to wild-type (FIG. 8).These results show satellite cell proliferation is delayed in α7integrin null muscle after cardiotoxin-induced injury.

To examine if the developmental program regulating muscle repair wasaffected by the loss of the α7β1 integrin, expression of Pax7 and MyoD(FIGS. 9 & 10) was examined. Pax7 is expressed in both quiescent andactivated satellite cells, while MyoD is expressed only indifferentiated myoblasts. Compared to wild-type muscle, α7 integrin nullmice exhibited 2- to 3-fold fewer Pax7 positive cells compared towild-type at 4 and 10 days post-cardiotoxin injury (FIG. 9). By day 28,similar numbers of Pax7 positive cells were observed in wild-type and α7integrin null mice.

Analysis of MyoD expression showed α7 integrin null muscle contained 28and 50-fold fewer MyoD positive myoblasts compared to wild-type muscleat 4 and 10 days post-cardiotoxin induced damage, respectively (FIG.10). By day 28 similar numbers of MyoD positive cells were observed inwild-type and α7 integrin null mice.

Together these results indicate loss of the α7 integrin results in feweractivated satellite cells in injured skeletal muscle and a delayedresponse in the developmental program that regulates myogenicdifferentiation.

Laminin Treatment of α7 Integrin Null Mice

Laminin-111 Treatment Restores Sarcolemmal Integrity in α7 Integrin NullMice

Recent studies have shown loss of the α7 integrin results in reducedlaminin expression. To explore if reduced laminin deposition couldaccount for the defective muscle regenerative phenotype observed in α7integrin null mice, the TA muscle was injected with laminin-111 threedays prior to cardiotoxin injury.

Laminin-111 was injected into the TA muscle of 2 week old wild-type andα7 integrin null pups and tissue analyzed by immunofluorescence using ananti-laminin-α1 antibody. Muscle injected with PBS alone contained nolaminin-111. By day 4, laminin-111 was abundantly in the extracellularmatrix surrounding muscle fibers and persisted for more than 28 days.Titration of laminin-111 in cultured myofibers revealed increasedtoxicity at concentrations 200 nM and higher (data not shown).

Surprisingly, the injected laminin-111 rapidly permeated the entire TAmuscle within 24-72 hours (FIG. 11 and supplemental data) and wasmaintained throughout the muscle for at least 31 days (FIG. 11). At alltime points after cardiotoxin-injury, α7 integrin null muscle treatedwith laminin-111 externally appeared identical to wild-type muscle (FIG.12).

Analysis of EBD uptake after cardiotoxin-induced injury revealed nodifference in the percentage of EBD-positive myofibers betweenlaminin-treated wild-type or α7 integrin null muscle at all time points(FIG. 13). These results demonstrate that injection of laminin-111 priorto cardiotoxin-induced injury restored sarcolemmal integrity to α7integrin null muscle.

Laminin Mediated Muscle Regeneration in Integrin Null Mice

To examine the ability of laminin-111 to improve muscle regeneration,5-week-old wild-type and α7 integrin null TA muscle were injected withlaminin and subjected to cardiotoxin-induced injury. Muscle sectionswere stained with H&E and mononuclear cell infiltrate and centrallylocated nuclei examined (FIG. 14). No difference in the myofiber size,centrally located nuclei or mononuclear cell infiltrate was observed inwild-type and α7 integrin null muscle treated with laminin-111 at 4, 10or 28 days following injury (FIG. 14).

Quantitation of centrally located nuclei confirmed that laminin-111treatment restored muscle regeneration to wild-type levels (FIG. 15). Atall time points, analyzed percentages of centrally located nuclei inlaminin-treated wild-type and α7 integrin null mice were notsignificantly different from each other. These results indicate thatlaminin-111 restored muscle repair in α7 integrin null muscle towild-type levels.

The ability of laminin-111 to restore regenerative capacity to α7integrin null muscle was examined by assaying eMyHC expression (FIG.16). At day 0, 7.3% of wild-type fibers and 9.7% of α7 integrin nullfibers were eMyHC positive as a result of injection with laminin (FIG.16). At 4 and 10 days after cardiotoxin treatment, wild-type and α7integrin null muscle exhibited similar levels of eMyHC expression (FIG.16). At 28 days post-injury, eMyHC was only present in negligibleamounts in the wild-type muscle while 34.4% of myofibers in α7 integrinnull muscle were positive for eMyHC (FIG. 16). These results demonstrateinjection of laminin-111 greatly improved the regenerative capacity ofα7 integrin null muscle.

Laminin Therapy Restores Myofiber Area in α7 Integrin Null Mice

Myofiber cross-sectional area was examined in laminin-treated wild-typeand α7 integrin null mice before and after cardiotoxin-induced injury(FIG. 17). At 4 days post-injury, the cross-sectional area of musclemyofibers in wild-type mice was found to be only 13% larger compared toα7 integrin deficient muscle (FIG. 17). By days 10 and 28post-cardiotoxin injury, the cross-sectional area of myofibers in α7integrin null muscle was similar to wild-type animals (FIG. 17).Together these data indicate treatment with laminin-111 restored musclerepair and myofiber size in α7 integrin null muscle.

Laminin Promotes Satellite Cell Proliferation in α7 Integrin NullInjured Muscle

To examine if laminin treatment improved satellite cell proliferation,BrdU incorporation after muscle injury was measured (FIG. 18). At 0, 4and 10 days post-cardiotoxin injury, no difference was observed in thenumber of BrdU positive satellite cells in wild-type and α7 integrinnull muscle (FIG. 18). At 28 days post-injury, there were significantlymore BrdU positive satellite cells in α7 integrin muscle comparedwild-type (FIG. 18). These results indicate treatment with lamininrestored satellite cell proliferation to wild-type levels.

Laminin Treatment Restores Myoblast Differentiation to α7 Integrin NullMuscle

To examine if treatment with laminin-111 restored the myogenic repairprogram in α7 integrin null muscle, expression of Pax7 and MyoD wasexamined (FIGS. 19 & 20). Prior to cardiotoxin-injury, wild-type and α7integrin-null muscle exhibit a few Pax7 positive cells, which could beattributed to the minor damage from the laminin injection (FIG. 19). At4 days post-cardiotoxin injury, there were 20% fewer Pax7 positive cellsin laminin-treated α7 integrin null muscle compared to wild-type (FIG.19). At 10 and 28 days post-cardiotoxin injury levels of Pax7 positivecells in the α7 integrin null muscle were similar to wild-type muscle(FIG. 19).

Analysis of MyoD revealed a few positive cells in laminin-treatedwild-type and α7 integrin null TA muscle at day 0 (FIG. 20). At days 4and 10 post-cardiotoxin injury, the number of MyoD positive cells inlaminin-treated α7 integrin null muscle was approximately 20-25% lowerthan wild-type (FIG. 20). However by day 28, both wild-type and α7integrin null muscle had similar numbers of MyoD positive cells (FIG.20). These data indicate that laminin treatment substantially restoredthe number of myogenic cells and promoted activation of the myogenicprogram involved in muscle repair in α7 integrin null muscle.

Discussion

This Example demonstrates that α7 integrin null mice exhibit defectiveskeletal muscle regeneration after cardiotoxin-induced injury. Treatmentwith laminin corrected the defective repair phenotype. Although someaspects of the myogenic developmental program have been elucidatedduring skeletal muscle regeneration, the mechanisms by which theextracellular matrix and integrin cell surface receptors participate inmyogenic repair are generally not well understood.

Muscle damage is followed by the rapid activation of satellite cells.Upon activation, these cells proliferate and activate myogenicdevelopmental programs to repair damaged muscle. Models suggest asubpopulation of satellite cells remain as stem cells to replace cellsthat have progressed down the myogenic lineage pathway. Duringactivation satellite cells express the transcription factors Pax3, Pax7,MyoD, myogenin and MRF4.

This Example demonstrates that loss of the α7 integrin leads to reducedsatellite cell proliferation as determined by reduced BrdU incorporationand Pax7 expression in cardiotoxin-treated α7 integrin null muscle. Inaddition, myoblast differentiation was significantly reduced in injuredα7 integrin deficient muscle as measured by MyoD expression. These dataindicate the α7β1 integrin regulates a key transition early in muscleregeneration in which satellite cells are activated to proliferate anddifferentiate into myogenic cells capable of repairing muscle.

Results presented in this Example demonstrate a significant reduction inthe presence of centrally located nuclei and delay in the expressioneMyHC in injured α7 integrin null myofibers. The presence of centrallylocated nuclei and expression of eMyHC suggest that α7 integrindeficient myoblasts are capable of fusion in vivo. These observationssupport in vitro studies which demonstrate primary α7 integrin nullmyoblasts can fuse to form myotubes in cell culture. Together theseobservations suggest the delay in muscle repair in vivo is primarily dueto defects in myoblast proliferation and differentiation leading tofewer myogenic cells capable of repairing damaged muscle.

Since the regenerative capacity of skeletal muscle is dependant on anintricate interplay between satellite cells and the extracellularmatrix, absence of the α7 integrin may result in loss of an optimallaminin-rich microenvironment required for myogenic repair. To determineif decreased laminin deposition contributes to the reduced muscleregenerative phenotype observed in α7 integrin null mice, laminin-111was injected into the muscle of mice prior to injury. Laminin isnormally produced by skeletal muscle cells and secreted into thesurrounding basal lamina. Interestingly, within 48-72 hours injectedlaminin-111 protein spread throughout the entire TA muscle and persistedfor more than 31 days in the basal lamina. Injection of muscle withlaminin-111 protein prior to cardiotoxin-induced injury restored muscleregeneration in α7 integrin null mice to wild-type levels. These datademonstrate that loss of the laminin microenvironment in α7integrin-deficient skeletal muscle is the underlying cause of the defectin muscle repair observed in these animals.

While laminin-211 and laminin-221 are expressed in adult muscle,laminin-111 is only present in embryonic skeletal muscle. One possibleexplanation for the improved muscle regeneration in laminin-treated α7integrin null muscle is that injection of laminin-111 may recapitulatean embryonic myogenic program in adult skeletal muscle. Activation ofthis embryonic program may result in enhanced myoblast activation andproliferation and improved muscle repair. However injection oflaminin-111 into wild-type skeletal muscle did not increase regenerativecapacity suggesting laminin-111 acted to replace laminin-211/221 in α7integrin deficient skeletal muscle. These results suggest other lamininreceptors are expressed in satellite cells that normally interact withlaminin to promote myogenic repair or can act to compensate for the lossof α7 integrin in myoblasts.

This Example suggests that subjects with α7 integrin mutations sufferfrom congenital myopathy as a result of reduced muscle regenerativecapacity due to reduced laminin-211/221 deposition. These data alsodemonstrate that direct injection of purified laminin-111 protein mayserve as a potential therapy for patients with α7 integrin-congenitalmyopathy. Since loss of regenerative capacity has been implicated in avariety of muscular dystrophies including MDC1A and DMD, laminin-111protein therapy may be beneficial in other forms of muscular dystrophy.

Example 2 Materials and Methods

Animals

C57BL/10ScSn (wild-type) and C57BL/10ScSn-Dmdmdx/J (mdx) strains of mice(Jackson Laboratories, Bar Harbor, Me.) were used in these studies inaccordance with an animal protocol approved by the University of Nevada,Reno Animal Care and Use Committee.

Isolation of α7β-gal^(+/−) Myoblasts

The gastrocnemius muscles were removed from 10-day-old α7βgal^(+/−) miceand tissue minced with scissors. Cells were enzymatically dissociatedwith 1.25 mg/ml collagen type II (Worthington Biochemical Corporation,Lakewood, N.J.) for 1 h at 37° C. The slurry was gently triturated andfiltered through nylon mesh. Cells were separated from muscle fiberfragments by differential centrifugation and plated onto 100 mm tissueculture plates. Myoblasts were maintained in proliferation medium(Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetalbovine serum (FBS), 0.5% chick embryo extract, 1% L-glutamine and 1%penicillin/streptomycin).

β-Galactosidase Staining

Myoblasts or myotubes were fixed in 4% paraformaldehyde for 5 minutes,washed with 1×PBS and permeabilized with a sodium deoxycholate/NP40mixture for 30 minutes. X-gal (50 mM potassium ferrocyanide, 50 mMpotassium ferricyanide, 1 M MgCl₂, and 100 mg/ml X-gal) was added to theplates and incubated at 37° C. for 2 hours. Plates were washed in PBS.Images were captured with a dissecting microscope and Spot digitalcamera.

Fluorescence Activated Flow Sorting (FACS)

Approximately 1×10⁶ α7βgal^(+/−) myoblasts were seeded on 100 mm cellculture plates coated with 0.1% gelatin and incubated overnight at 37°C. Growth media was removed and cells were treated for 16-24 hours with100 nM LAM-111 in PBS. Cells were trypsinized, counted, pelleted and 30μl DMEM containing 20% FBS growth medium added. 30 μl of 200 nM of FDG(Molecular Probes, Eugene, Oreg.) was added to the cells and incubatedat 37° C. for 1 minute. To stop the reaction 600 μl of ice cold growthmedia was added to each sample and incubated on ice for 20 minutes.Samples were run on the Beckman Coulter XL/MCI flow cytometer andanalyzed using FlowJo software.

Laminin-111 Injections

Natural mouse laminin (Invitrogen) at 100 nM in PBS was injected intothe left tibialis anterior (TA) muscle of 10 day mdx mice. Thecontralateral right TA muscle was injected with PBS and served as acontrol. Mice were sacrificed and muscle was harvested at 5 weeks ofage. For systemic delivery 1 mg/kg of laminin-111 in PBS was injectedintraperitoneally at 10 days and tissues harvested for analysis at 5weeks of age. Control mdx mice were injected with the same volume ofPBS.

Evan's Blue Dye Uptake

Mice were injected intraperitoneally with 50 μl 10 g of body weight withsterile Evans blue dye solution (10 mg/ml). After 3 hours, the TA musclewas harvested and flash-frozen in liquid nitrogen, 10 μm cryosectionswere placed on microscope slides and fixed in 4% paraformaldehyde. Tooutline muscle fibers, tissue sections were incubated with 2 μg/mlOregon Green-488 conjugated wheat germ aggluttinn (WGA) (MolecularProbes, Eugene, Oreg.). A minimum of 1000 fibers per animal were countedto determine the percentage of muscle fibers positive for Evans blue dyeuptake. At least five animals from each genotype were analyzed. Imageswere captured and counted at 630× magnification.

Blood Chemistry

Blood was collected at 5 weeks of age and allowed to clot at roomtemperature for a minimum of 30 minutes. After centrifugation at 3000rpm, serum was collected. Serum was sent to Comparative PathologyLaboratory at the University of California, Davis to assay for creatinekinase, creatine and blood urea nitrogen (BUN).

Immunofluorescence

Tissues were embedded in Tissue-TEK Optimal Cutting Temperature compound(Sakura Finetek USA Inc., Torrance, Calif.). Using a Leica CM1850cryostat (Leica Microsystems, Wetzlar, Del.), 10-micron sections wereplaced onto Surgipath microscope slides (Surgipath Medical Industries,Richmond, Ill.). The α7 integrin was detected with a 1:1000 dilution ofanti-CA5.5 rat monoclonal antibody (Sierra Biosource, Morgan Hill,Calif.) followed by a 1:1000 dilution of FITC-conjugated anti-ratsecondary antibody. The β1D integrin was detected with a 1:500 dilutionof rabbit polyclonal antibody followed by a 1:500 dilution ofFITC-conjugated anti-rabbit antibody. Laminin-al was detected with a1:500 dilution of MAB1903 (Chemicon International, Temecula, Calif.).Dystrophin was detected with the mouse monoclonal Dys2 antibody(Novacastra Laboratories, Ltd, Newcastle upon Tyne, UK) and utrophin wasdetected with MANCHO7 7F3 monoclonal antibody against utrophin (GlennMorris, Center for Inherited Neuromuscular Disease, Shropshire, UK) at adilution of 1:200. The mouse monoclonal antibodies were used inconjunction with a mouse-on-mouse (MOM) immunodetection kit (VectorLaboratories, Burlingame, Calif.) to block mouse immunoglobulin and a1:500 dilution of FITC-conjugated anti-mouse secondary antibody.Acetylcholine receptors were detected with Rhodamine-labeledα-bungarotoxin at 1:1000 (Molecular Probes, Eugene, Oreg.). Fluorescencewas observed with a Zeiss Axioskop 2 Plus fluorescent microscope andimages were captured with Zeiss AxioCam HRc digital camera andAxiovision 4.1 software (all available from Carl Zeiss MicroImaging,Thornwood, N.Y.).

Histology

Tissue sections were fixed in ice-cold 95% ethanol for 2 minutesfollowed by 70% ethanol for 2 minutes and then re-hydrated in runningwater for 5 minutes. The sections were stained with Gill's hematoxylin(Fisher Scientific, Fair Lawn, N.J.) and rinsed in water for 5 minutes.Sections were placed in Scott's solution (0.024 M NaHCO3, 0.17 M MgSO4)for 3 minutes and rinsed in water for 5 minutes. Sections were thenstained with eosin (Sigma-Aldrich, St Louis, Mo.) for 2 minutes.Sections were progressively dehydrated in ice-cold 70% and 95% ethanolfor 30 seconds each, followed by 100% ethanol for 2 minutes and clearedin xylene for 5 minutes prior to mounting with DePeX mounting medium(Electron Microscopy Sciences, Washington, Pa.). Central myonuclei inregenerating muscles were counted at 630× magnification by bright-fieldmicroscopy. The number of central nuclei per muscle fiber was determinedby counting a minimum of 1000 muscle fibers per animal. At least fiveanimals from each genotype were analyzed.

Immunoblotting

To analyze α7 integrin, protein was extracted using 200 mMoctyl-β-D-glucopyranoside (Sigma Aldrich, St Louis, Mo.), 50 mM Tris-HClpH 7.4, 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 2 mM PMSF and a 1:200dilution of Protease Inhibitor Cocktail Set III (Calbiochem, EMDBiosciences, San Diego, Calif.). Lysate was collected and centrifugedfor 15 minutes at 10,000×g, and supernatant was transferred to a freshtube. Protein was quantified by Bradford assay and 40 μg of totalprotein was separated on 7.5% SDS-PAGE gels under non-reducedconditions, and transferred to nitrocellulose membranes. Membranes wereblocked in Odyssey Blocking Buffer (LiCor Biosciences, Lincoln, Nebr.)that was diluted 1:1 in phosphate-buffered saline (PBS). The α7 integrinwas detected with a 1:500 dilution of rabbit anti-α7B (B2 347)polyclonal antibody. Blots were incubated with a 1:5000 dilution ofAlexa Fluor 680-coupled goat anti-rabbit IgG (Molecular Probes, Eugene,Oreg.) to detect the primary antibody.

To examine utrophin expression, protein was extracted from the PBS andLAM-111 injected mdx and wild-type tibialis anterior muscle with RIPAbuffer (50 mM Hepes pH 7.4, 150 mM NaCl, 1 mM Na₃VO₄, 10 mM NaF, 0.5%Triton X-100, 0.5% NP40, 10% glycerol, 2 mM PMSF and a 1:200 dilution ofProtease Inhibitor Cocktail Set III) and quantified by Bradford assay(BioRad Laboratories Inc., Hercules, Calif.). 80 μg of total proteinwere separated on a 7.5% SDS-PAGE gel and transferred to nitrocellulosemembrane. The blot was incubated with a 1:200 dilution of anti-utrophinmouse monoclonal antibody (MANCHO3 8A4, a kind gift of Glenn Morris,Center for Inherited Neuromuscular Disease, Shropshire, UK) followed bya 1:50,000 dilution of horseradish peroxidase (HRP)-labeled goatanti-mouse secondary antibody. The 395 kDa utrophin band was detected bychemiluminescence and normalized for protein loading by probing the sameblot with anti-Cox-1 antibody (Santa Cruz Biotechnology, Santa Cruz,Calif.). Band intensities were quantified by using ImageQuant TLsoftware (Amersham Biosciences, Piscataway, N.J.).

Statistical Analysis

All averaged data are reported as the mean±standard deviation.Comparisons between multiple groups were performed by one-way-analysisof variance (ANOVA) for parametric data or by Kruskal-Wallisone-way-analysis of variance on ranks for non-parametric data usingSigmaStat 1.0 software (Jandel Corporation, San Rafael, Calif.). P<0.05was considered statistically significant.

Discussion

Duchenne Muscular Dystrophy (DMD) is a devastating neuromuscular diseasecaused by mutations in the gene encoding dystrophin. The α7β1 integrinand utrophin are laminin binding proteins up-regulated in the muscle ofDMD patients and in the mdx mouse model. Transgenic over-expression ofutrophin or α7 integrin in dystrophic mice alleviates muscle diseasemaking these genes targets for pharmacological intervention. Todetermine whether laminin regulates α7 integrin expression, culturedmouse and human myoblasts were treated with laminin and assayed for α7integrin expression. This Example demonstrates that laminin-111, a formof laminin highly expressed during embryonic development, increased α7integrin expression in cultured myoblasts from mice and DMD patients.Intramuscular injection of laminin-111 into mdx mice increased β7integrin and utrophin expression, stabilized the sarcolemma andprevented muscle pathology. Systemic laminin-111 protein therapyrestored serum creatine kinase levels in mdx mice to the normal range.These findings demonstrate laminin-111 is a highly potent and novelprotein therapeutic for the mouse model of DMD and represents a novelparadigm for the systemic delivery of extracellular matrix proteins as atherapy for genetic diseases.

Duchenne Muscular Dystrophy (DMD) is the most common X-linked diseaseaffecting 1 in 3,500 male births. DMD patients exhibit severe andprogressive muscle wasting with symptoms first detected at 2 to 5 yearsof age. As the disease progresses, patients are confined to wheelchairs,require ventilator assistance and die in their second or third decade oflife. To date there is no effective treatment or cure for thisdevastating neuromuscular disease.

DMD patients and mdx mice (the mouse model for DMD) have mutations inthe gene encoding dystrophinm, resulting in a loss of dystrophinprotein. Dystrophin is a 427 kDa protein located on the innercytoplasmic membrane of muscle fibers. Through its N-terminal rod domainrepeats, dystrophin interacts with F-actin of the cell cytoskeleton. TheC-terminal region of dystrophin interacts with a transmembrane complexcomposed of α- and β-dystroglycans, dystrobrevins, α- and β-syntrophinsand sarcoglycans. The dystrophin glycoprotein complex provides atransmembrane linkage between the cell cytoskeleton and laminin in theextracellular matrix of muscle. Loss of dystrophin results in a failureof this critical laminin-binding complex to form, leading to damage andprogressive muscle weakness.

In the absence of dystrophin, two additional laminin-binding complexes,the α7β1 integrin and utrophin glycoprotein complexes, are up-regulatedin the skeletal muscle of DMD patients and mdx mice. Transgenicenhancement of utrophin or α7 integrin in skeletal muscle alleviatesmuscle disease in dystrophic mice. On the other hand, loss of utrophinor α7 integrin in mdx mice results in more severe phenotypes and reducedviability. Together these results indicate that utrophin and the α7β1integrin are genetic modifiers of disease progression and targets fordrug-based therapies that boost their expression.

To whether particular molecules increase α7 integrin expression, amuscle cell based assay was developed. A α7 integrin null mouse wasproduced in which exon 1 of the gene encoding the α7 integrin wasreplaced by the LacZ reporter gene. In these mice, all thetranscriptional regulatory elements are retained allowing α7 integrinpromoter activity to be reported by β-galactosidase. Primary myoblasts(designated α7βgal^(+/−)) were isolated from 10 day old α7^(+/−) pups.α7βgal^(+/−) myoblasts expressed β-galactosidase which increased upondifferentiation (FIGS. 21 and 22), consistent with the expressionpattern of α7 integrin in myoblasts and myotubes. The activity of the α7integrin promoter was measured by β-galactosidase cleavage of thenon-fluorescent compound fluorescein di-β-D-galactopyranoside (FDG) tofluorescein.

Several lines of evidence suggest positive feedback in the regulation oflaminin and α7 integrin expression. Mutations in the gene encodinglaminin-α2 result in congenital muscular dystrophy type 1A (MDC1A). BothMDC1A patients and laminin-α2 deficient mice have dramatically reducedlevels of α7 integrin which may contribute to severe muscle pathology.In addition, laminin-α2 is decreased in α7 integrin null skeletalmuscle. To determine the relationship between laminin and α7 integrinexpression, α7βgal^(+/−) myoblasts were exposed to variousconcentrations of laminin-111 from 0-200 nM for 24 hours. Studiessuggest laminin-111 is functionally similar to laminin-211 and interactswith the α7β1 integrin. Fluorescence activated cell sorting (FACS)analysis revealed maximal α7 integrin promoter activity at 100 nMlaminin-111 (FIG. 23).

α7 integrin protein in laminin-111 treated C2C12 mouse myoblasts and DMDprimary myoblasts was quantified. Protein extracts from laminin-treatedmyoblasts were subjected to Western analysis to detect α7B integrin.Laminin-111 produced a 2-fold increase in α7B integrin in C2C12 and DMDmyoblasts (FIGS. 24 and 25). These data indicate laminin-111 increasesα7 integrin expression in human and mouse muscle cells.

It was then determined whether the above in vitro results withlaminin-111 could be translated in vivo to increase α7 integrinexpression in skeletal muscle. The left tibialis anterior (TA) musclesof 10 day old mdx mice were injected with 100 μl of 100 nM laminin-111,while the right TA muscle was injected with 100 μl PBS and served as thecontralateral control. At 5 weeks of age mice were sacrificed and the TAmuscles were harvested. Laminin-111 is not normally expressed in adultmuscle and the injected protein was detected with an anti-laminin-α1antibody. Immunofluorescence revealed the injected laminin-111 proteinwas deposited throughout the basal lamina of the TA muscle of 5 week oldmdx mice (FIG. 26). The images also confirm that dystrophin was presentin the wild type muscle, but absent in both the PBS and laminin-111treated mdx muscle.

To determine if laminin-111 prevented muscle pathology in mdx mice,Evans blue dye (EBD) uptake and hemotoxylin and eosin (H&E) stainingwere performed on cryosections from PBS and laminin-111-injected TAmuscle (FIG. 27). Analysis revealed that mdx muscles injected withlaminin-111 had a 12-fold reduction in the percentage of fibers positivefor EBD compared to the contralateral controls (FIG. 28, *P<0.05,**P<0.001, n=5 mice/group). In addition, mdx muscles injected withlaminin-111 showed a 4-fold decrease in the percentage of muscle fiberswith centrally located nuclei (FIG. 28, *P<0.05, **P<0.001, n=5mice/group). These results indicate laminin-111 protein therapydramatically increased sacrolemmal integrity and reduced the requirementfor muscle repair.

To determine the mechanism by which laminin-111 protein therapyprotected dystrophin-deficient muscle from damage, immunofluorescenceanalysis of utrophin and α7 integrin were done. Results revealedincreased expression and extrajunctional localization of α7 integrin andutrophin in laminin-111-treated muscles of mdx mice compared to controls(FIG. 29).

To confirm and quantify these observations, PBS and laminin-111-treatedmdx muscles were subjected to Western analysis (FIG. 30). A 1.6- and2.6-fold increase in α7A and α7B integrin isoforms respectively wasobserved in laminin-111 treated mdx muscles compared with controls (FIG.31, *P=<0.05, **P=<0.001, n=5 mice/group). Protein loading wasnormalized to cyclooxygenase-1 (cox-1). In addition, a 1.3-fold increasein utrophin was observed in laminin-111-treated muscles (FIG. 31,*P=<0.05, **P=<0.001, n=5 mice/group). No significant change in β1Dintegrin levels was seen, consistent with results reported in α7integrin transgenic mice. These results indicate that laminin-111increased the expression of both α7 integrin and utrophin, two proteinsknown to alleviate muscle pathology when transgenically over-expressedin dystrophic muscle.

DMD patients suffer from generalized muscle wasting. An effectivetherapy therefore should target all muscles, including the heart anddiaphragm. It was then determined if laminin-111 protein could bedelivered systemically to these muscles. Ten day-old mdx pups wereinjected intraperitoneally with one dose of laminin-111 at 1 mg/kg andtissues analyzed at 5 weeks of age. Immunofluorescence analysis revealedthe presence of laminin-α1 throughout the basal lamina of diaphragm andgastrocnemius muscles of laminin-111 injected mice, while controls werenegative (FIGS. 32 and 33). Analysis of cardiac muscle showedlaminin-111 surrounding cardiomyocytes (FIG. 32).

To determine if systemic delivery of laminin-111 was therapeutic, serumwas collected 3 weeks after laminin-111 injections and creatine kinaselevels measured. Serum creatine kinase is highly elevated in DMDpatients due to muscle damage, and levels of creatine kinase are usedfor diagnostic and prognostic purposes. This Example demonstrates thatlaminin-111 therapy resulted in a 2.6-fold reduction in serum creatinekinase levels compared to PBS control (FIG. 34, *P<0.05, n=5mice/group). These levels were not statistically different from creatinekinase levels in wild-type mice. These results demonstrate thatlaminin-111 protein can be systemically delivered to major musclesystems in mdx mice to prevent dystrophic pathology.

Since the laminin-111 protein is relatively large and could adverselyaffect renal function, serum creatine and blood urea nitrogen (BUN) weremeasured. Analysis revealed that creatine and BUN were not statisticallydifferent between laminin-111-treated mdx and control mice (FIG. 34,*P<0.05, n=5 mice/group). These data indicate laminin-111 proteintherapy had no adverse effects on renal function.

This Example demonstrates for the first time that a single systemic doseof laminin-111 protein prevents the onset of muscle disease for at leastthree-weeks in mice genetically destined to develop muscular dystrophy.Together these findings demonstrate that laminin-111 may be a highlypotent, novel protein therapeutic for Duchenne Muscular Dystrophy. Inaddition, laminin-111 protein therapy may prove effective in thetreatment of other muscle diseases including congenital musculardystrophy type 1A, Limb-Girdle muscular dystrophy and α7 integrincongenital myopathy. The effectiveness of laminin-111 protein injectionsin dystrophic mice represents a novel paradigm demonstrating thatsystemic delivery of extracellular matrix proteins could be explored asa therapy for genetic diseases.

It is to be understood that the above discussion provides a detaileddescription of various embodiments. The above descriptions will enablethose skilled in the art to make many departures from the particularexamples described above to provide apparatuses constructed inaccordance with the present disclosure. The embodiments areillustrative, and not intended to limit the scope of the presentdisclosure. The scope of the present disclosure is rather to bedetermined by the scope of the claims as issued and equivalents thereto.

1. A method of enhancing muscle regeneration, repair, or maintenance ina subject comprising administering a therapeutically effective amount ofat least a portion of a laminin to the subject.
 2. The method of claim1, wherein the laminin comprises at least a portion of laminin-1.
 3. Themethod of claim 1, wherein the laminin comprises laminin-1.
 4. Themethod of claim 1, where the laminin comprises all or a portion oflaminin-1, laminin-2, laminin-4, or combinations thereof.
 5. The methodof claim 1, wherein the laminin comprises the a1 chain of laminin. 6.The method of claim 1, wherein the laminin comprises at least a portionof the a1 chain of laminin.
 7. The method of claim 1, further comprisingdiagnosing the subject with a condition characterized by impaired muscleregeneration prior to administering a therapeutically effective amountof at least a portion of a laminin to the subject.
 8. The method ofclaim 1, wherein the subject has a condition characterized by impairedproduction of a component of the costamere.
 9. The method of claim 1,wherein the subject has impaired production of dystrophin.
 10. Themethod of claim 1, wherein the subject has impaired production oflaminin.
 11. The method of claim 1, wherein the subject has impairedproduction of a7pi integrin.
 12. The method of claim 1, wherein thelaminin is administered with an additional therapeutic agent.
 13. Themethod of claim 1, wherein the additional therapeutic agent is selectedfrom a costameric protein, a growth factor, satellite cells, stem cells,and myocytes.
 14. The method of claim 1, wherein the laminin isadministered prior to the subject experiencing muscle damage or disease.15. The method of claim 1, wherein the laminin is administeredsystemically to the subject.
 16. The method of claim 1, wherein thelaminin is selected from a derivative, analog, or fragment of a laminin.17. The method of claim 1, wherein the laminin is administered in aconcentration of between about 50 nM and about 200 nm.
 18. The method ofclaim 1, wherein the laminin is administered in an amount between about1 nmol/g and about 50 nmol/g of the subject's weight.
 19. A method ofpromoting wound healing in a subject comprising administering aneffective amount of laminin to the subject.
 20. A method ofprospectively preventing or reducing muscle injury or damage in asubject comprising administering an effective amount of laminin to thesubject.